The ubiquitously expressed kinase GAK (cyclin G-associated kinase, also known as auxillin 2) is a 160 kDa serine/threonine protein kinase that was first identified as a cyclin G1-binding protein. GAK is composed of a N-terminal kinase domain that phosphorylates the μ-subunits of adaptor proteins 1 and 2, a clathrin binding domain, and a C-terminal J-domain that interacts with a heat shock cognate 70 (Hsc70).
As suggested by its strong homology (43%) to the neuronal-specific protein auxilin, a heat shock cognate 70 (Hsc70) cochaperone with a role in uncoating clathrin vesicles, GAK regulates clathrin-mediated membrane trafficking as an essential cofactor for the Hsc70-dependent uncoating of clathrin-coated vesicles. Moreover, down-regulation of GAK by a small hairpin RNA enhanced the levels of expression and tyrosine kinase activity of EGFR and altered the spectrum of downstream signaling, at least partly due to alterations in receptor trafficking.
GAK forms a complex with Cyclin G and the protein phosphatase 2A (PP2A) B′γ subunit, which suggests that it may play yet unidentified roles in cellular events other than membrane trafficking. In support of this hypothesis, GAK acts as a transcriptional coactivator of the androgen receptor (AR; a ligand-dependent transcription factor), and GAK expression was significantly increased in hormone refractory prostate cancer. Moreover, both GAK and its association partner clathrin heavy chain (CHC), localize to both the cytoplasm and nucleus with distinct association modes, and CHC colocalizes with GAK in the nucleus, while Cyclin G and PP2A B′γ are also present in the nucleus.
Moreover, siRNA-mediated GAK knockdown caused cell-cycle arrest at metaphase, which revealed two novel functions of GAK: maintenance of proper centrosome stability and of mitotic chromosome congression.
High-throughput screening of the kinome is a powerful tool with which one can identify multiple kinases related to the survival of cancer cells. Kinase shRNA screening revealed that the loss of function of GAK, among others, resulted in marked growth inhibition of osteosarcoma cells (Mol. Cancer Ther. 2010, 9 (12), 3342-3350).
In contrast to the high expression of GAK in osteosarcomas, normal human osteoblasts expressed only low quantity of the protein. The result of kinase shRNA screening was further confirmed by siRNA knockdown of GAK on several osteosarcoma cell lines. Although 100 nmol/L of nonspecific siRNA did not have any cytotoxic activity on osteosarcoma cell lines, a concentration of as low as 10 nmol/L of GAK siRNA was enough to inhibit the proliferation of osteosarcoma cells. Importantly, it had similar effects on both drug-sensitive and drug-resistant osteosarcoma cell lines, which implicate that it exerts its effects independently of ATP-binding cassette transporters such as P-gp. This was further confirmed by Western blot analysis of P-gp, which did not show any effect on Pgp trafficking. Therefore, GAK has the potential to be a target for the treatment of drug-naive osteosarcomas as well as multidrug-resistant osteosarcomas.
In addition, recent genome wide association studies (GWAS) have been performed to identify genetic risk factors in sporadic, non-familial forms of disease. The GWAS showed an association for a few genes previously identified in the linkage studies (alpha-synuclein, LRRK2) and also identified new genetic risk factors for Parkinson's disease, such as glucocerebrosidase (GBA), microtubule associated protein tau (MAPT), PARK16, the human leukocyte antigen (HLA) locus, bone marrow stromal antigen 1 (BST1) and GAK (The Application of Clinical Genetics 2011, 4, 67-80). GAK has been labeled as the PARK17 gene. Later studies confirmed an association between Parkinson's disease and GAK (Hum. Genet. 2009, 124, 593-605; Nature Genet. 2010, 42, 781-785; Hum. Mol. Genet. 2011, 20, 345-353; Neurology 2012, 79, 659-667; Ann. Hum. Genet. 2011, 75, 195-200). A systematic meta-analysis in Parkinson's disease genetics confirms overall probability values showing robust association of genetic markers in the GAK gene with Parkinson's disease (PLoS Genet. 2012, 8, e1002548). Therefore, multiple genetic studies confirm a role for GAK in Parkinson's disease.
Hypoxia induces changes to cancer cells that facilitate their survival, make them more resistant to classical drug treatments and increases the metastatic potential of tumor cells. Researchers from Wyeth have therefore carried out a kinome wide siRNA screening in order to identify kinase genes that affect hypoxic colon cancer cells (J Biomol Screen. 2013 18, 782-796.). Hits identified in the screen were characterized for effects on different molecular responses to hypoxia. The hits were validated by short hairpin RNA studies. These studies led to the observation that GAK plays in important role in the adaptation of cancer cells to hypoxia. Therefore, GAK can be considered as a promising drug target for the treatment of cancer cells within the hypoxic regions of a solid tumor.
GAK phosphorylates the μ-subunits of adaptor proteins 1 and 2, which are membrane trafficking proteins. AP2M1, the μ subunit of AP-2, has recently been shown to be essential for Hepatitis C virus (HCV) assembly. While its knockdown had no effect on HCV RNA replication, it dramatically decreased both intra- and extra-cellular infectivity, consistent with an assembly defect (Plos Pathogens 2012, 8, e1002845). Phosphorylation of AP2M1 by GAK stimulates binding of AP2M1 to the core protein of HCV. Hence, GAK is a cellular host factor essential in mediating HCV assembly. Kinase inhibitors, such as erlotinib, which is known to target GAK, inhibits AP2M1 phosphorylation, disrupts the binding of core to AP2M1 and inhibits HCV assembly, as well as infectious virus production. Therefore, GAK inhibitors hold the potential to treat HCV infections, based on inhibition of a viral-host interaction.
The synthesis of a very limited number of isothiazolo[4,3-b]pyridine has been described in literature. The synthesis of 3-aminoisothiazolo[4,3-b]pyridine from 3-aminopicolinonitrile via 3-aminothiopicolinamide, followed by a subsequent oxidative cyclization with H2C2 to give 3-amino-isothiazolo[4,3-b]pyridine has been described in Can. J. Chem. 1973, 51(11), 1741-1748. Isothiazolo[4,3-b]pyridines have also been synthesized also as M1 positive allosteric modulators (Bioorg. Med. Chem. Lett. 2010, 20, 2533-2537).
However, none of these documents teaches or suggests isothiazolo[4,3-b]pyridine derivatives having the substitution pattern disclosed by the present invention nor their use as antiviral medicaments.
However, there is a continuous need in the art for specific and highly therapeutically active compounds, that act as GAK inhibitors and are useful for the treatment of viral infections.